天津医药

天津医药 ›› 2019, Vol. 47 ›› Issue (4): 382-386.doi: 10.11958/20181840

• 细胞与分子生物学 • 上一篇    下一篇

苯乙双胍通过影响线粒体功能促进A549/DDP细胞凋亡

侯永旺，常娇，王艳辉，任丽   

  1. 天津医科大学肿瘤医院，国家肿瘤临床医学研究中心，天津市“肿瘤防治”重点实验室，天津市恶性肿瘤临床医学研究中心（邮 编300060）
  • 收稿日期:2018-11-22 修回日期:2019-02-26 出版日期:2019-04-15 发布日期:2019-05-27
  • 通讯作者: 任丽 E-mail:renlitianjin@163.com
  • 作者简介:侯永旺（1990），男，硕士在读，主要从事肿瘤代谢方面研究

Phenformin promotes A549/DDP cell apoptosis by affecting mitochondrial function

HOU Yong-wang, CHANG Jiao, WANG Yan-hui, REN Li   

  1. Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Tianjin Key Laboratory of Cancer Prevention and Therapy, Tianjin Clinical Research Center for Cancer, Tianjin 300060, China
  • Received:2018-11-22 Revised:2019-02-26 Published:2019-04-15 Online:2019-05-27
  • Contact: Li REN E-mail:renlitianjin@163.com

PDF (PC)

3653

摘要： 目的 探究苯乙双胍抑制 A549/DDP细胞增殖并促进其凋亡的可能机制。方法 MTS法检测顺铂（顺铂 组）和苯乙双胍（苯乙双胍组）对 A549/DDP 细胞和 A549 细胞的细胞活力（24 h 和 48 h）的影响。苯乙双胍组以 2.5 mmol/L苯乙双胍处理，对照组采用PBS处理，MTS法检测苯乙双胍对A549/DDP细胞增殖的影响，克隆形成实验检测 苯乙双胍对 A549/DDP细胞克隆形成能力的影响，流式细胞术检测苯乙双胍对 A549/DDP细胞凋亡和活性氧（ROS） 胞内线粒体质量（Mitotracker）、钙离子（Rhod-2）的影响，实时荧光定量PCR检测线粒体DNA（mtDNA）变化，三磷酸腺 苷（ATP）试剂盒检测 ATP的变化，Western blot检测苯乙双胍对线粒体氧化磷酸化复合物表达的影响。结果 与顺 铂组比较，苯乙双胍组A549/DDP细胞在24 h和48 h的细胞活力降低（P＜0.05），而2组间A549细胞24 h和48 h活力 差异无统计学意义。顺铂处理48 h时A549/DDP细胞活力均高于A549细胞（P＜0.05），而24 h间差异无统计学意义； 苯乙双胍处理的A549/DDP细胞活力低于A549细胞（P＜0.05）。与PBS组相比，苯乙双胍组A549/DDP细胞的24 h和 48 h 增殖率、集落形成数减低，而凋亡率升高，ROS 水平增高，Mitotracker、Rhod-2、mtDNA 及 ATP 水平降低（P＜ 0.05），线粒体氧化磷酸化复合物Ⅰ中的NDUFB8蛋白、复合物Ⅱ中的SDHB蛋白和复合物Ⅳ中的MTCO1蛋白表达明 显降低（P＜0.05）。结论 （1）A549/DDP细胞较 A549细胞对苯乙双胍更为敏感，但对顺铂耐受性较 A549细胞强。 （2）苯乙双胍通过增加细胞内 ROS、减少线粒体质量、钙离子和 mtDNA、抑制线粒体氧化磷酸化复合物表达，减少 ATP生成，从而抑制A549/DDP细胞增殖并促进其凋亡。

关键词: 苯乙双胍, 线粒体, A549/DDP细胞

Abstract: Objective To investigate the possible mechanism of phenformin inhibiting proliferation and promoting apoptosis of A549/DDP cells. Methods MTS assay was used to detect the cell viability of A549/DDP cells treated with cisplatin and phenformin. The treatment group was treated with phenformin (2.5 mmol/L), and control group was treated with phosphate buffer saline (PBS). MTS assay was used to detect the effect of phenformin on the proliferation of A549/DDP cells. The colony formation ability was detected by colony formation assay. Flow cytometry was used to detect the effects of phenformin on apoptosis, reactive oxygen species (ROS), mitochondrial mass (Mitotracker) and calcium ion (Rhod-2) in A549/DDP cells. The real-time quantitative PCR was used to detect mitochondrial DNA (mtDNA), and ATP was detected by ATP kit. The effect of phenformin on mitochondrial oxidative phosphorylation complex was detected by Western blot assay. Results Compared with the cisplatin group, the cell viability ratios of A549/DDP cells were decreased at 24 h and 48 h in the phenformin group (P＜0.05), but there was no significant difference in the viability at 24 h and 48 h of A549 cells between the two groups. The viability of A549/DDP cells treated with cisplatin 48 h was high compared with that of A549 cells, but there was no significant difference in the viability of 24 h. The viability of A549/DDP cells treated with phenformin was significantly lower than that of A549 cells (P＜0.05). Compared with the PBS group, the proliferation rate and colony formation of A549/DDP cells decreased at 24 h and 48 h in the phenformin group, while the apoptosis rate and the ROS level increased (P＜0.05). Mitotracker, Rhod-2, mtDNA and ATP were also decreased (P＜0.05). The NDUFB8 protein in complex I, the SDHB protein in complex II, and the MTCO1 protein in complex IV were significantly decreased (P＜0.05). Conclusion (1) A549/DDP cells are more sensitive to phenformin than A549 cells, but more resistant to cisplatin than A549 cells. (2) Phenformin inhibits proliferation and promots apoptosis of A549/DDP cells by increasing intracellular ROS, decreasing mitochondrial mass, calcium ion and mtDNA and inhibiting mitochondrial oxidative phosphorylation complex, and reducing ATP production.

Key words: phenformin, mitochondria, A549/DDP cell